This invention relates to a method for the production of urokinase.
Urokinase is an enzyme capable of converting plasminogen into plasmin. As such, it has been found useful as an activator for promoting the lysis of blood clots.
Urokinase occurs in small amounts in human urine, but it has been calculated that isolation of the enzyme from that source requires at least 1500 liters of urine per clinical dose of the enzyme.
More recently, urokinase has been obtained from cultures of kidney cells from various animals such as, e.g. mouse kidney, baby hamster kidney and, in particular, human embryonic kidney (HEK) cells. It has been shown that urokinase isolated from HEK cells grown in tissue culture is identical to urokinase from urine. Barlow and Lazer, Thromb. Res. 1 (3), 201-7 (1972).
Various methods have been suggested heretofore to improve the production of urokinase by cell culture techniques. According to one such method described in U.S. Pat. No. 3,904,480, various antimitotic agents are employed in the cell culture medium to increase the yield of plasminogen activator (urokinase). In another approach to the problem, the kidney cell culture is overlayed with fibrin to increase the urokinase yield. Bernik and Kwaan, J. Lab. Clin. Med. 70, 650 (1967); J. Clin Invest. 48, 1740 (1969); J. Clin. Invest. 52, 823 (1973). Other materials heretofore suggested for addition to the cell culture medium in specified amounts for increasing the yield of urokinase are pronase, as described in U.S. Pat. No. 3,930,944, and glycine, as taught in U.S. Pat. No. 3,930,945.
Still another method for improving the production of urokinase involves selection of cells that produce the enzyme and elimination of non-producers, as described by Barlow et al., "Production of Plasminogen Activator by Tissue Culture Techniques", in "Proteases and Biological Control", Cold Spring Harbor Conference on Cell Proliferation, Vol. 2, 1975, Cold Spring Harbor Lab. (eds. Reich, Rifkin and Shaw), at pages 325-31. The latter publication also indicates that the addition of extra amounts of amino acids such as aspartic acid and glutamic acid do not increase the production of plasminogen activator but that addition of plasminogen and other proteases did increase the yield. U.S. Pat. No. 3,930,940 similarly teaches that amino acids other than glycine (none specified) have no or sometimes opposite effects on urokinase production.